Limits...
Expression of the CTCF-paralogous cancer-testis gene, brother of the regulator of imprinted sites (BORIS), is regulated by three alternative promoters modulated by CpG methylation and by CTCF and p53 transcription factors.

Renaud S, Pugacheva EM, Delgado MD, Braunschweig R, Abdullaev Z, Loukinov D, Benhattar J, Lobanenkov V - Nucleic Acids Res. (2007)

Bottom Line: We found that DNA methylation and functional p53 contributes to the negative regulation of each promoter.Moreover, reduction of CTCF in normally BORIS-negative human fibroblasts resulted in derepression of BORIS promoters.These results provide a mechanistic basis for understanding cancer-related associations between haploinsufficiency of CTCF and BORIS derepression, and between the lack of functional p53 and aberrant activation of BORIS.

View Article: PubMed Central - PubMed

Affiliation: Section of Molecular Pathology, Laboratory of Immunopathology, NIAID, NIH, Rockville, MD 20815, USA.

ABSTRACT
BORIS, like other members of the 'cancer/testis antigen' family, is normally expressed in testicular germ cells and repressed in somatic cells, but is aberrantly activated in cancers. To understand regulatory mechanisms governing human BORIS expression, we characterized its 5'-flanking region. Using 5' RACE, we identified three promoters, designated A, B and C, corresponding to transcription start sites at -1447, -899 and -658 bp upstream of the first ATG. Alternative promoter usage generated at least five alternatively spliced BORIS mRNAs with different half-lives determined by varying 5'-UTRs. In normal testis, BORIS is transcribed from all three promoters, but 84% of the 30 cancer cell lines tested used only promoter(s) A and/or C while the others utilized primarily promoters B and C. The differences in promoter usage between normal and cancer cells suggested that they were subject to differential regulation. We found that DNA methylation and functional p53 contributes to the negative regulation of each promoter. Moreover, reduction of CTCF in normally BORIS-negative human fibroblasts resulted in derepression of BORIS promoters. These results provide a mechanistic basis for understanding cancer-related associations between haploinsufficiency of CTCF and BORIS derepression, and between the lack of functional p53 and aberrant activation of BORIS.

Show MeSH

Related in: MedlinePlus

Identification and activity of three BORIS promoters. (A) Nucleotide sequence of the 5′-upstream region of the human BORIS gene is shown together with a portion of the coding region. Potential binding sites of transcriptional regulatory proteins predicted by the MatInspector program are shown in boxes. Yellow boxes highlight the Sp1 factor. The A in ATG translational start site is designated as nucleotide +1 and does not appear in the figure. The main start sites for transcription, as determined by 5′-RACE, are indicated by encircled blue arrows. The transcribed sequences are underlined. Nucleotides of the CpG island are shaded in green. Red numbers (1–32) represent all CpGs within the CpG island. (B) Determination of BORIS promoters A, B and C activities by transient expression of luciferase reporter constructs in HeLa, NCCIT and OVCAR-3 cell lines. Left: schematic representation of BORIS promoter luciferase reporter constructs. Numbers indicate positions of nucleotides of the 5′-flanking region of the BORIS gene, as shown in A. Right: relative luciferase activities compared with the pGL3 control vector activity, which was considered to have 100% activity. The CpG island is shaded in green.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2175345&req=5

Figure 1: Identification and activity of three BORIS promoters. (A) Nucleotide sequence of the 5′-upstream region of the human BORIS gene is shown together with a portion of the coding region. Potential binding sites of transcriptional regulatory proteins predicted by the MatInspector program are shown in boxes. Yellow boxes highlight the Sp1 factor. The A in ATG translational start site is designated as nucleotide +1 and does not appear in the figure. The main start sites for transcription, as determined by 5′-RACE, are indicated by encircled blue arrows. The transcribed sequences are underlined. Nucleotides of the CpG island are shaded in green. Red numbers (1–32) represent all CpGs within the CpG island. (B) Determination of BORIS promoters A, B and C activities by transient expression of luciferase reporter constructs in HeLa, NCCIT and OVCAR-3 cell lines. Left: schematic representation of BORIS promoter luciferase reporter constructs. Numbers indicate positions of nucleotides of the 5′-flanking region of the BORIS gene, as shown in A. Right: relative luciferase activities compared with the pGL3 control vector activity, which was considered to have 100% activity. The CpG island is shaded in green.

Mentions: To identify transcription initiation sites, 5′ RLM-RACE was performed. The 5′ RLM-RACE method has a major advantage over other methods for mapping of transcription start sites (e.g. primer extension, nuclease protection assays or traditional 5′ RACE) in that it only detects authentic capped 5′ ends of mRNAs. The technique is based on RNA ligase-mediated (RLM-RACE) and oligo-capping rapid amplification of cDNA ends, and results in the selective ligation of an RNA oligonucleotide to the 5′ ends of decapped mRNA. This method allowed the amplification of only truly full-length transcripts via elimination of truncated messages. The 5′ RLM-RACE assays were performed on total RNA extracted from normal testis tissue, and from the NCCIT tumor cell line. Two PCR products (∼700 bp and ∼1500 bp) were obtained and, after cloning and sequencing, the first two start sites were identified at −658 and −899 bp from the ATG translational start site (Figure 1A). Using the GC-Rich PCR system and nested primers upstream of the first transcription start site, a ∼200 bp PCR product was generated. This new site mapped to −1447 bp upstream of the ATG translation start codon (Figure 1A).Figure 1.


Expression of the CTCF-paralogous cancer-testis gene, brother of the regulator of imprinted sites (BORIS), is regulated by three alternative promoters modulated by CpG methylation and by CTCF and p53 transcription factors.

Renaud S, Pugacheva EM, Delgado MD, Braunschweig R, Abdullaev Z, Loukinov D, Benhattar J, Lobanenkov V - Nucleic Acids Res. (2007)

Identification and activity of three BORIS promoters. (A) Nucleotide sequence of the 5′-upstream region of the human BORIS gene is shown together with a portion of the coding region. Potential binding sites of transcriptional regulatory proteins predicted by the MatInspector program are shown in boxes. Yellow boxes highlight the Sp1 factor. The A in ATG translational start site is designated as nucleotide +1 and does not appear in the figure. The main start sites for transcription, as determined by 5′-RACE, are indicated by encircled blue arrows. The transcribed sequences are underlined. Nucleotides of the CpG island are shaded in green. Red numbers (1–32) represent all CpGs within the CpG island. (B) Determination of BORIS promoters A, B and C activities by transient expression of luciferase reporter constructs in HeLa, NCCIT and OVCAR-3 cell lines. Left: schematic representation of BORIS promoter luciferase reporter constructs. Numbers indicate positions of nucleotides of the 5′-flanking region of the BORIS gene, as shown in A. Right: relative luciferase activities compared with the pGL3 control vector activity, which was considered to have 100% activity. The CpG island is shaded in green.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2175345&req=5

Figure 1: Identification and activity of three BORIS promoters. (A) Nucleotide sequence of the 5′-upstream region of the human BORIS gene is shown together with a portion of the coding region. Potential binding sites of transcriptional regulatory proteins predicted by the MatInspector program are shown in boxes. Yellow boxes highlight the Sp1 factor. The A in ATG translational start site is designated as nucleotide +1 and does not appear in the figure. The main start sites for transcription, as determined by 5′-RACE, are indicated by encircled blue arrows. The transcribed sequences are underlined. Nucleotides of the CpG island are shaded in green. Red numbers (1–32) represent all CpGs within the CpG island. (B) Determination of BORIS promoters A, B and C activities by transient expression of luciferase reporter constructs in HeLa, NCCIT and OVCAR-3 cell lines. Left: schematic representation of BORIS promoter luciferase reporter constructs. Numbers indicate positions of nucleotides of the 5′-flanking region of the BORIS gene, as shown in A. Right: relative luciferase activities compared with the pGL3 control vector activity, which was considered to have 100% activity. The CpG island is shaded in green.
Mentions: To identify transcription initiation sites, 5′ RLM-RACE was performed. The 5′ RLM-RACE method has a major advantage over other methods for mapping of transcription start sites (e.g. primer extension, nuclease protection assays or traditional 5′ RACE) in that it only detects authentic capped 5′ ends of mRNAs. The technique is based on RNA ligase-mediated (RLM-RACE) and oligo-capping rapid amplification of cDNA ends, and results in the selective ligation of an RNA oligonucleotide to the 5′ ends of decapped mRNA. This method allowed the amplification of only truly full-length transcripts via elimination of truncated messages. The 5′ RLM-RACE assays were performed on total RNA extracted from normal testis tissue, and from the NCCIT tumor cell line. Two PCR products (∼700 bp and ∼1500 bp) were obtained and, after cloning and sequencing, the first two start sites were identified at −658 and −899 bp from the ATG translational start site (Figure 1A). Using the GC-Rich PCR system and nested primers upstream of the first transcription start site, a ∼200 bp PCR product was generated. This new site mapped to −1447 bp upstream of the ATG translation start codon (Figure 1A).Figure 1.

Bottom Line: We found that DNA methylation and functional p53 contributes to the negative regulation of each promoter.Moreover, reduction of CTCF in normally BORIS-negative human fibroblasts resulted in derepression of BORIS promoters.These results provide a mechanistic basis for understanding cancer-related associations between haploinsufficiency of CTCF and BORIS derepression, and between the lack of functional p53 and aberrant activation of BORIS.

View Article: PubMed Central - PubMed

Affiliation: Section of Molecular Pathology, Laboratory of Immunopathology, NIAID, NIH, Rockville, MD 20815, USA.

ABSTRACT
BORIS, like other members of the 'cancer/testis antigen' family, is normally expressed in testicular germ cells and repressed in somatic cells, but is aberrantly activated in cancers. To understand regulatory mechanisms governing human BORIS expression, we characterized its 5'-flanking region. Using 5' RACE, we identified three promoters, designated A, B and C, corresponding to transcription start sites at -1447, -899 and -658 bp upstream of the first ATG. Alternative promoter usage generated at least five alternatively spliced BORIS mRNAs with different half-lives determined by varying 5'-UTRs. In normal testis, BORIS is transcribed from all three promoters, but 84% of the 30 cancer cell lines tested used only promoter(s) A and/or C while the others utilized primarily promoters B and C. The differences in promoter usage between normal and cancer cells suggested that they were subject to differential regulation. We found that DNA methylation and functional p53 contributes to the negative regulation of each promoter. Moreover, reduction of CTCF in normally BORIS-negative human fibroblasts resulted in derepression of BORIS promoters. These results provide a mechanistic basis for understanding cancer-related associations between haploinsufficiency of CTCF and BORIS derepression, and between the lack of functional p53 and aberrant activation of BORIS.

Show MeSH
Related in: MedlinePlus